Protein carbohydrates interactions play crucial role in biology for e.g. cell adhesion, cell recognition, immunoassay and fertilization. These biological events involve multivalent binding of the ligand to the host. The importance of carbohydrates in biologically relevant recognition processes has only recently come to light. (Feizi, et al., Biochem. J. 245:1, 1987; Belvilacqua, et al., Science 243:1160, 1989). They demonstrated carbohydrates, along with proteins and nucleic acids, act as primary biological information carriers.
Relatively few investigations are reported despite known role of carbohydrates in biology, for enhancing such interactions. Various targets for carbohydrate such as enzymes, proteins and viruses are being identified which can have numerous applications in therapeutics. Rouhi, A. M., (Chem. Engg. News, Sept. 23, 62–66, 1996) reported critical role of carbohydrates in various biological processes such as cell recognition, cell adhesion, cell differentiation, inflammation, viral and bacterial infection, tumerigenesis, and metastasis.
One of the major advantages of carbohydrate modification may be that it can impart change in physical characteristics such as solubility, stability, activity, antibody recognition and susceptibility to enzyme. Sharon, et al. (Science 246:227–234, 1989) suggested carbohydrate portions of glyco-conjugate molecules to be an important entity in biology.
Carbohydrates can be incorporated in polymer chain and utilized for binding to the receptors. Thereby, the polymers can be coupled with the other polymers containing ligands for multivalent effect.
Deng, S. J., and Narang, S. A. (Proc. Natl. Acad. Sci. USA, 92, 4992–4996, 1995) studied improved carbohydrate-binding single-chain antibodies from synthetic gene libraries. The dimeric antibodies have faster on-rates than the monomeric parent antibodies.
Stahl, et al. (U.S. Pat. No. 6,037,467, 2000) reported methods for preparing hydrophilic polymers by coupling the carbohydrate portion to the hydrophilic polymer portion.
Recent patent granted to Krepinsky, et al (U.S. Pat. No. 6,184,368, 2001) suggests the application of carbohydrates in preventing the infections. Mandeville, et al. (U.S. Pat. No. 5,891,862, 1999 and U.S. Pat. No. 6,187,762, 2001) reported the use of polyvalent polymers containing carbohydrates for the treatment of rotavirus infection.
There is a necessity to prepare multivalent ligands for enhanced binding as monovalent ligands display weak affinities and poor specificity towards the receptor binding sites. The resultant saccharide in a multivalent form can bind to the same substrate with greater affinity and specificity. The binding of cell surface receptors to multivalent carbohydrate molecules exhibits wide variety of biological responses and has unique edge over monovalent interactions (Mammen, et al. Angew. Chem., Int. Ed., 37, 2754–2794, 1998). Laura Kiessling and Nicola L. Pohl reported (Chemistry & Biology, 3:71–77, 1996) newer structural templates for the generation of multivalent carbohydrates containing multivalent saccharide derivatives useful for biological recognition events.
Synthetic multivalent moieties can be prepared with recognition of binding host sites, moreover they can be structured with molecular flexibility and orientation around the host. The characteristics of multivalent interactions are different than their monovalent counterparts as the latter involve one to one binding whereas multivalent interaction involves simultaneous binding of ligands at multiple sites of host molecules.
Many chemical and chemoenzymetic methods have been reported for the preparation of di- and trivalent ligands, dendrimers, and high molecular weight polymers, but involve complex synthetic methods. Thus, there is a need to devise simple methodology to obtain multivalent ligands of varying polymolecularity.
Polymers comprising multiple ligands could be more effective inhibitors for the host cell receptor, as a result of higher affinity for the pathogen. In addition, the higher molecular weight of the polymeric ligands also prevents the infection through steric exclusion. (Spaltenstein, A., and Whitesides, G. M., J. Am. Chem. Soc., 113, 686, 687, 1991).
Agglutination of erythrocytes caused by influenza virus can be prevented by use of polyvalent sialic acid inhibitors. This novel approach which is a model for pathogen-host interactions was reported by Mammen, M., and Whitesides, G., M., (J. Med. Chem. 38:21, 4179–90, 1995). The authors reported polymers containing sialic acid as effective inhibitors of influenza virus. Moreover, they suggested two favorable mechanisms for inhibition between the surfaces of virus and erythrocytes 1) High-affinity binding through polyvalency, and 2) Steric stabilization.
Sigal et al. (J. Am. Chem. Soc., 118:16, 3789–3800, 1996) studied the efficacy of polymers containing sialoside groups in inhibiting the adhesion of influenza virus to erythrocytes. They delineated the contributions of enhanced substrate ligand binding and steric considerations to efficiency of inhibition. These investigators reported sialic acid ligands, which can be exploited for the inhibition of the influenza virus. Monomeric inhibitor requires a higher concentration for inhibition since they are required to occupy at least half of the sialic acid binding sites on the virus, whereas the high molecular weight inhibitors need only a few attachments to achieve the same.
Dimick et al. (J. Am. Chem. Soc., Vol. 121 No 44, 10286, 1999) reported the molecular cluster glycoside effects and the synthesis of polyvalent ligands for the plant lectin concanavalin A.
Various methods have been reported in the past to synthesize multivalent ligands such as ring-opening metathesis polymerization (ROMP). ROMP has been used to generate well defined, biologically active polymers by Gibson et al., (Chem. Comm., 1095–1096, 1997) and Biagini et al., (Polymer, 39, 1007–1014, 1998).
A number of researchers have reported the synthesis and evaluation of sialoside-containing polyacrylamide inhibitors of the influenza virus. Whitesides and coworkers Mammen, M., Dahmann, G. & Whitesides, G. M. (J. Med. Chem. 38, 4179–4190, 1995) demonstrated effective inhibitors of hemagglutination by influenza virus synthesized from polymers comprising active ester groups. They used a broad range of sialic acid substituted acrylamide copolymers to probe the mechanism of inhibition of hemagglutination by multivalent carbohydrates.
Choi, S. K., Mammen, M. & Whitesides, G. M. (Chemistry & Biology, 3, 97–104, 1996) demonstrated the hemagglutination activity of monomeric sialic acid towards the influenza neuramimidase is considerably enhanced when the sialic acid is conjugated with a polymer so that it is presented as a multiple sialosides.
An understanding of the mode of action of the polyvalent sialosides provides a method for the design of inhibitors for influenza virus and insights into the mechanisms through which natural polyvalent ligands might act.
Carbohydrate-conjugated polymers have been based mostly on the polyacrylamide backbone. Alternative polymer in the backbone may be more effective. The effect of methods for synthesis of the saccharide-modified materials on their inhibition efficiency may be attributed to the density of functional groups.
Recently, the ring-opening metathesis polymerization (ROMP) methods have been applied for the synthesis of carbohydrate-substituted materials (Mortell, K. H., Gingras, M. & Kiessling, L. L. (J. Am. Chem. Soc. 116, 12053–12054, 1994). Like acrylamide polymerization, ROMP can be used in polar solvents and the carbohydrate residues need not be protected. Jason E. Gestwicki, Laura E. Strong, Christopher W. Cairo, L., Frederick J. Boehm, and Laura L. Kiessling, Chemistry & Biology, Vol. 9, 163–169, 2002, demonstrated the use of polymers generated by ring-opening metathesis polymerization (ROMP) as scaffolds to noncovalently assemble multiple copies of a lectin, the tetravalent protein concanavalin A (Con A).
The synergetic application of stimuli-responsive polymers and interactive molecules to form site-specific conjugates useful in variety of assays, separations, processing, and other uses are disclosed by Hoffman; Allan S.; Stayton; Patrick, S. (U.S. Pat. No. 5,998,588, 1999). The interactive molecules used can be biomolecules such as polysaccharides or glycoproteins, proteins or peptides, as antibodies, receptors, or enzymes, which specifically bind to ligands in the suitable environment. The inventors prepared stimuli-responsive polymers coupled to the recognition biomolecules at a specific site so that the polymer can be manipulated by stimulation to alter ligand-biomolecule binding at an adjacent binding site, for example, the biotin binding site of streptavidin, the antigen-binding site of an antibody or the active, substrate-binding site of an enzyme.
It is very important that ligand which is conjugated to polymers binds to active site of biomolecule must also be evicted from the binding site with change in environment. Such polymer conjugates find application in selective phase separation or affinity precipitation of biomolecules. The polymers used for such applications can be stimulus-responsive to an appropriate environmental stimulus.
Many chemical and chemoenzymatic routes of synthesizing multivalent ligands have been adapted for the preparation of di- and trivalent ligands, (Glick, G. D.; Toogood, P. L.; Wiley: D. C.; Skehel, J. J.; Knowles, J. R., J. Biol. Chem. 1991, 266, 23660–23669) dendrimers, and high molecular weight polymers, (Choi, S. K.; Mammen, M.; Whitesides, G. M. (J. Am. Chem. Soc., 119, 4103–4111, 1997) but well defined, linear oligomers synthesis in the past are complicated and requires multiple steps.
Thus, methods of synthesizing block copolymers with defined multivalent ligands for enhanced interactions provide a means for exploring biologically important processes.
Christopher W. Cairo, Jason E. Gestwicki, Motomu Kanai, and Laura L. Kiessling, J. Am. Chem. Soc. 124: 8,1614–1618, 2002) analyzed three aspects of receptor clustering: the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Their experiments reveal that the density of binding sites on a multivalent ligand strongly influence binding characteristics. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, are the most efficient on a binding epitope. Moreover, results provide insight into the design of ligands for controlling receptor-receptor interactions, which mimic mechanisms by which natural multivalent ligands bind to the substrates.
Damschroder et al. (U.S. Pat. No. 2,548,520, 1951) disclosed high molecular weight materials prepared by copolymerizing proteins conjugated with unsaturated monomers or proteins conjugated with preformed polymers. Synthesis of these high molecular weight materials generally requires temperatures up to 100° C. Such high temperatures are not well tolerated by most of the proteins. Thus the methods described are unsuitable for producing polymers of biologically active molecules.
Jaworek, et al. (U.S. Pat. No. 3,969,287, 1976) reports a method for the preparation of carrier-bound proteins, wherein the protein is reacted with a monomer containing at least one double bond capable of copolymerization. The carrier is provided as a water-insoluble solid or is produced in situ by the polymerization of water-soluble monomers in the presence of the protein monomer conjugate. The proteins utilized in the method of this invention are typically enzymes.
The carbohydrates such as NAG serve as ligands for lectins and lysozyme. Roy et al. (J. Chem. Soc. Chem. Comm., 1611–1613, 1992) reported custom designed glycopolymer synthesis by terpolymerizations. The N-acryloyl precursors and the acrylamide were used as effector molecules to provide specific properties such as hydrophobicity and mimicking tyrosine residues of proteins.
Mochalova et al. (Antiviral Research, 23,179–190, 1994) reported carbohydrate inhibitors like sialic acid anchored to polymeric or liposomal carriers. They conjugated glycylamido benzylsialoside with poly (acrylic acid-co-acrylamides) and dextrans. These polymeric ligands were evaluated for their ability to bind influenza A and B virus strains in cell culture.
Nishimura (Macromolecules 27, 4876–4880, 1994) demonstrated the inhibitory effect of glycosylated-cyclodextrins on the erythrocytes agglutination induced by wheat germ (Triticum vulgaris) agglutinin was observed at 240-fold lower concentration than its monomeric counterpart.
Dimick et al. (J. Am. Chem. Soc., 121, 44, 10286, 1999) explored newer strategies based on enhancing interactions. Synthesis of polyvalent ligands was reported and the role of glycosidic clusters in enhancing binding with plant lectin concanavalin A was demonstrated.
In an alternative approach Kanai, et al. (J. Am. Chem. Soc., 119, 9931–9932, 1997) reported ring opening metathesis polymerization (ROMP) consisting multivalent mannose binding to concanavalin A. However the methods are complicated and do not control “living” nature of glycopolymer.
Yamada et al. (Macromolecules, 32,3553–3558, 1999) reported controlled synthesis of amphiphilic block co polymers with pendant N-Acetyl Glucosamine (NAG) residues by living cationic polymerization. Copolymer architecture resulted in an enhancement in binding between Wheat Germ Agglutinin (WGA) and NAG.
Hoffman et al. (J. Biomater. Sci., Polym. Ed., 4:5, 545, 1993) synthesized carboxyl terminated poly (N-isopropyl acrylamide) oligomers, which were reacted with biopolymers to form thermo-reversible polymer-enzyme conjugates.
Krepinsky, et al. (U.S. Pat. No. 6,184,368, 2001) reported methods for synthesis of polyvalent carbohydrate molecules by glycosylations of partially protected polysaccharides bearing a single glycosylating agent or a mixture of glycosylating agents. The patent explains the non-productive binding of chitosan to lysozyme.
Chitosan (Formula 4) is linear, binary heteropolysaccharide and consists of 2-acetaamido-2-deoxy-β-D-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-D-glucose (GlcNAc, D-unit). The active site of lysozyme comprises subsites designated A–F. Specific binding of chitosan sequences to lysozyme begins with binding of the NAG units in the subsite C. Moreover natural ligands derived from glucose are susceptible to microbial growth. There is need to synthesize ligands similar to repeat units of chitosan which will not be hydrolyzed by lysozyme. These polymers are expected to be more stable than chitin and chitosan (Formula 4).

Apart from the type of the ligand, its distribution along the polymer chain also plays a crucial role in influencing the efficiency of the inhibition.